Targeting LIN28B reprograms tumor glucose metabolism and acidic microenvironment to suppress cancer stemness and metastasis.

The altered metabolism and acidic microenvironment plays an important role in promoting tumor malignant characteristics. A small population of cancer stem cells (CSCs) were considered as a therapy target to reserve tumor relapse, resistance, and metastasis. However, the molecular mechanism that regulates CSCs metabolism remains poorly understood. In this study, we demonstrate a fundamental role of stemness gene LIN28B in maintaining CSCs glycolysis metabolism. Using LIN28B-expressing cancer cell lines, we found that the rate of extracellular acidification, glucose uptake, and lactate secretion are all suppressed by LIN28B knockdown in vitro and in vivo. Importantly, metabolic analyses reveal that CSCs have enhanced aerobic glycolysis metabolic characteristics and the glycolytic product lactate further promotes cancer associated stemness properties. LIN28B silencing suppresses MYC expression that further increases miR-34a-5p level. Furthermore, the glycolysis metabolism of human breast cancer cell line MDA-MB-231 is suppressed by either MYC siRNA or miR-34a-5p mimic. Clinically, high MYC and low miR-34a-5p level are correlated with high LIN28B expression and poor prognosis in human breast cancer patients. Notably, blocking LIN28B/MYC/miR-34a-5p signaling pathway by LIN28B-specific inhibitor causes dramatic inhibition of tumor growth and metastasis in immunodeficient orthotopic mouse models of human breast cancer cell MDA-MB-231. Taken together, our findings offer a preclinical investigation of targeting LIN28B to suppress CSCs glycolysis metabolism and tumor progression that may improve the therapeutic benefit for cancer patients.

miR-100 maintains phenotype of tumor-associated macrophages by targeting mTOR to promote tumor metastasis via Stat5a/IL-1ra pathway in mouse breast cancer.

Tumor-associated macrophages (TAMs), the main part of immune cells in tumor microenvironment (TME), play a potent role in promoting tumorigenesis through mechanisms such as stimulating angiogenesis, enhancing tumor migration and suppressing antitumor immunity. MicroRNAs (miRNAs) are considered as crucial regulators in multiple biological processes. The relationship between miRNAs and macrophages function has been extensively reported, but the roles that miRNAs play in regulating TAMs phenotype remain unclear. In this study, we screened highly expressed microRNAs in TAMs, and first identified that miR-100 represented a TAMs-high expression pattern and maintained TAMs phenotype by targeting mTOR signaling pathway. Moreover, miR-100 expression level in TAMs was positively related to IL-1ra secretion, a traditional immune-suppressive cytokine, which was determined to promote tumor cells stemness via stimulating Hedgehog pathway. Mechanism study suggested that mTOR/Stat5a pathway was involved in IL-1ra transcriptional regulation process mediated by miR-100. More importantly, tumor metastasis and invasion capacity were significantly decreased in a 4T1 mouse breast cancer model injected intratumorally with miR-100 antagomir, and combination therapy with cisplatin showed much better benefit. In this study, we confirm that highly expressed miR-100 maintains the phenotype of TAMs and promotes tumor metastasis via enhancing IL-1ra secretion. Interfering miR-100 expression of TAMs in mouse breast cancer model could inhibit TAMs pro-tumor function and reduce tumor metastasis, which suggests that miR-100 could serve as a potential therapy target to remodel tumor microenvironment in breast cancer.

Linc-RAM is required for FGF2 function in regulating myogenic cell differentiation.

Myogenic differentiation of skeletal muscle stem cells, also known satellite cells, is tightly orchestrated by extrinsic and intrinsic regulators. Basic fibroblast growth factor (FGF2) is well documented to be implicated in satellite cell self-renewal and differentiation by repressing MyoD. We recently identified a MyoD-regulated and skeletal muscle-specifically expressed long non-coding RNA Linc-RAM which enhances myogenic differentiation by facilitating MyoD/Baf60c/Brg1 complex assembly. Herein, we investigated the transcriptional regulation and intracellular signaling pathway in mediating Linc-RAM gene expression during muscle cell differentiation. Firstly, we demonstrate Linc-RAM is negatively regulated by FGF2 via Ras/Raf/Mek/Erk signaling pathway in muscle cells. Overexpression of MyoD significantly attenuates repression of Linc-RAM promoter activities in C2C12 cells treated with FGF2. Knockout of MyoD abolishes FGF2-mediated repression of Linc-RAM gene transcription in satellite cells sorted from skeletal muscle of MyoD-/-;Pax7-nGFP mice, suggesting inhibition of MyoD is required for FGF2-mediated expression of Linc-RAM. For the functional significance, we show that overexpression of Linc-RAM rescues FGF2-induced inhibition of C2C12 cell differentiation, indicating inhibition of Linc-RAM is required for FGF2-mediated suppression of myogenic differentiation. Consistently, we are able to further corroborate the requirement of Linc-RAM inhibition for FGF2-modulated repression of myogenic differentiation by using an ex vivo cultured single fiber system and satellite cells sorted from Linc-RAM-/-;Pax7-nGFP knockout mice. Collectively, the present study not only reveals the intracellular signaling in FGF2-mediated Linc-RAM gene expression but also demonstrate the functional significance of Linc-RAM in FGF2-mediated muscle cell differentiation.

NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells.

This study was conducted to determine the role of neuregulin 1 (NRG1)-dependent human epidermal growth factor receptor 3 (HER3) activation in trastuzumab primary resistance, and to observe the inhibitory effect of HER3 monoclonal antibody on HER2-overexpressing breast cancer cells. BT474 cells (trastuzumab sensitive) and MDA-MB-453 cells (trastuzumab resistant) were first stimulated with NRG1 and then treated with either trastuzumab, HER3 antibody, or a combination of both. The expression of phospho human epidermal growth factor receptor 2 (p-HER2), phospho human epidermal growth factor receptor 3 (p-HER3), phospho protein kinase B (p-Akt) and phospho mitogen-activated protein kinase (p-MAPK) were detected by western blotting. Apoptosis was detected by flow cytometry. Cell viability was detected by MTT assay. Without NRG1 stimulation, trastuzumab treatment significantly down-regulated the expression of p-HER2, increased early apoptosis, and decreased cell viability in BT474 cells. After NRG1 stimulation, the aforementioned effects weakened or disappeared in the trastuzumab treatment group, whereas in the HER3 antibody treatment group, there was significant downregulation in p-HER3 expression and increase in early apoptosis of BT474 cells. In MDA-MB-453 cells, the HER3 antibody significantly downregulated both p-HER2 and p-HER3 and promoted early apoptosis after NRG1 stimulation, however, trastuzumab hardly played a role. p-Akt and p-MAPK were also significantly downregulated by the HER3 antibody after NRG1 stimulation. The expressions of p-HER2, p-HER3, p-Akt and p-MAPK were all downregulated after HER3 gene silencing, compared to the control. NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells. HER3 monoclonal antibody combined with trastuzumab may serve as a treatment choice for patients with primary resistance to trastuzumab.

The anti-HER3 antibody in combination with trastuzumab exerts synergistic antitumor activity in HER2-positive gastric cancer.

The anti-HER2 monoclonal antibody trastuzumab is central to the treatment of HER2-positive gastric cancer (GC); however, its responses are limited. HER3 seems to be the preferred dimerization partner with HER2 and is emerging as a key target for complete blockade of downstream pathways and better clinical response. In this study, we report that novel anti-HER3 antibodies (1A5-3D4) that can neutralize multiple modes of HER3 activation, combined with trastuzumab, exhibited synergistic inhibitory effect on the cell proliferation in HER2-positive GC cell lines. Follow-up studies revealed that the combination treatment significantly inhibited phosphorylation of HER3 as well as AKT and ERK signals. In vivo experiments further showed that the anti-tumor effect of trastuzumab was enhanced by its combination with 1A5-3D4 in NCI-N87 xenograft and patient derived xenografts (PDX). Particularly in an HER2-negative whereas neuregulin1 (a ligand of HER3) positive PDX, the combination was also superior to monotherapy. 1A5-3D4 in combination with trastuzumab exhibits a synergistic inhibitory effect on tumor activity, suggesting that targeting both HER2 and HER3 resulted in an improved treatment effects on HER2-positive GC.

IKKß Enforces a LIN28B/TCF7L2 Positive Feedback Loop That Promotes Cancer Cell Stemness and Metastasis.

Considerable evidence suggests that proinflammatory pathways drive self-renewal of cancer stem-like cells (CSC), but the underlying mechanisms remain mainly undefined. Here we report that the let7 repressor LIN28B and its regulator IKBKB (IKKß) sustain cancer cell stemness by interacting with the Wnt/TCF7L2 (TCF4) signaling pathway to promote cancer progression. We found that LIN28B expression correlated with clinical progression and stemness marker expression in breast cancer patients. Functional studies demonstrated that the stemness properties of LIN28B-expressing human breast and lung cancer cells were enhanced by IKKß, whereas loss of LIN28B abolished stemness properties in these settings. These phenomena were driven through interactions with TCF7L2, which enhanced LIN28B expression by direct binding to intron 1 of the LIN28B gene, which in turn promoted TCF7L2 mRNA translation through a positive feedback loop. Notably, RNAi-mediated silencing of LIN28B or pharmacologic inhibition of IKKß was sufficient to suppress primary and metastatic tumor growth in vivo. Together, our results establish the LIN28B/TCF7L2 interaction loop as a central mediator of cancer stemness driven by proinflammatory processes during progression and metastasis, possibly offering a new therapeutic target for generalized interventions in advanced cancers.

Mechanisms for chemical transformations of (R,R)-tartaric acid on Cu(110): A first principles study.

Periodic density functional theory calculations are used to systematically investigate, for the first time, the mechanisms for chemical transformations of (R,R)-tartaric acid on a model Cu(110) surface. The overall potential energy surface for the chemical transformations is revealed. The calculations show that the adsorption of the intact biacid molecules of (R,R)-tartaric acid on Cu(110) surface is not strong, but upon adsorption on Cu(110), the biacid molecules will chemically transform immediately, rather than desorb from the surface. It is found that the chemical transformations of (R,R)-tartaric acid on Cu(110) is a thermodynamically favorable process, to produce the monotartrate species, bitartrate species, and H atoms. Kinetically, the initial reaction step is only one O-H bond scission in either one of the COOH group of a biacid molecule of (R,R)-tartaric acid leading to the formation of a monotartrate species and a H atom, which is an almost spontaneous process. The rate-controlling step is the O-H bond scission in the COOH group of a monotartrate species producing a bitartrate species and a H atom. The concerted reaction for simultaneously breaking the two O-H bonds in both COOH groups of a biacid molecule cannot proceed.

[Determination of Norfloxacin by its enhancement effect on the fluorescence intensity of functionalized CdS nanoparticles].

A novel assay of Norfloxacin with a sensitivity at the microgram level is proposed based on the measurement of enhanced fluorescence intensity signals by the interaction of functionalized nano-CdS with Norfloxacin. The CdS nanoparticles were synthesized by thioacetamide (TAA) and cadmium nitrate (Cd(NO3)2) in the alkaline solution, and the nanoparticles proved to be stable in the aqueous solution. At pH 7.4, the fluorescence signals of functionalized nano-CdS were greatly enhanced by Norfloxacin in the region of 300-700 nm characterized by the peak around 495 nm, and the surface defect-related emission located at 565 nm. However, the surface defect-related emission was unconspicuous, thus we concluded that the functionalized nano-CdS QDs (Quantum Dots) possessed excellent luminescence capability and favourable structure. At the same time, the absorption spectra and transmission electron microscopy (TEM) also proved this deduction. The external reaction conditions (such as effect of buffer system, pH, ionic strength, reaction time and temperature, colloid concentration) were discussed. The result showed that better fluorescence signals could be obtained in the condition of 0.10 mol x L(-1) Tris-HCl, pH 7.4, 0.1 mol x L(-1) NaCl and the reaction time 5 min. The fluorescence emission spectra of CdS QDs with increasing Norfloxacin concentration were recorded under the optimal condition. From the fluorescence intensity and peak position of nano-CdS colloidal as different concentration of Norfloxacin was added, the possible mechanism of reaction between mercapto-acetic acid capped CdS and Norfloxacin was discussed. Linear relationship can be established between the enhanced fluorescence intensity and Norfloxacin concentration in the range of 1.25-11.25 microg x mL(-1) (3.92-35.27 micromol x L(-1)) or 11.25-100.0 microg x mL(-1) (35.27-313.5 micromol x L(-1)). The limit of detection is 1.5 x 10(-3) x microg x mL(-1), which can be applied to the determination of blood serum samples. Based on this, a new direct quantitative determination method for Norfloxacin in synthetic samples without separation of foreign substances is established. At the same time, the possible enhancing mechanism is due to the formation of exciplex during reaction between nano-CdS and Norfloxacin, providing a guidance for the study of pharmacokinetics.

Nanometre-sized titanium dioxide-catalyzed reactions of nitric oxide with aliphatic cyclic and aromatic amines.

Activated on the surface of nanometre-sized TiO2, NO gas can easily react with aliphatic cyclic amines and aryl free radicals at RT under atmospheric pressure to offer NONOates and cupferron sodiums, respectively.